Cryo-EM structure of the human CST·Polα/Primase complex in a recruitment state

The CST·Polα/Primase complex is essential for telomere overhang maintenance and additionally functions to counteract resection at double-strand breaks. We report a 4.6-Å resolution cryo-EM structure of CST·Polα/Primase, captured prior to catalysis in a recruitment state, which provides insights into the architecture and stoichiometry of the fill-in machinery. Our model informs on human disease mutations that cause Coats plus syndrome.

Structural basis for the interaction of SARS-CoV-2 virulence factor nsp1 with Pol α - Primase

The molecular mechanisms that drive the infection by the SARS-CoV-2 coronavirus, the causative agent of the COVID-19 (Coronavirus disease-2019) pandemic, are under intense current scrutiny, to understand how the virus operates and to uncover ways in which the disease can be prevented or alleviated. Recent cell-based analyses of SARS-CoV-2 protein - protein interactions have mapped the human proteins targeted by the virus. The DNA polymerase α - primase complex or primosome, responsible for initiating DNA synthesis in genomic duplication, was identified as a target of nsp1 (non structural protein 1), a major virulence factor in the SARS-CoV-2 infection. Here, we report the biochemical characterisation of the interaction between nsp1 and the primosome and the cryoEM structure of the primosome - nsp1 complex. Our data provide a structural basis for the reported interaction between the primosome and nsp1. They suggest that Pol α - primase plays a part in the immune response to the viral infection, and that its targeting by SARS-CoV-2 aims to interfere with such function.

The three-component helicase/primase complex of herpes simplex virus-1

Herpes simplex virus type 1 (HSV-1) is one of the nine herpesviruses that infect humans. HSV-1 encodes seven proteins to replicate its genome in the hijacked human cell. Among these are the herpes virus DNA helicase and primase that are essential components of its replication machinery. In the HSV-1 replisome, the helicase–primase complex is composed of three components including UL5 (helicase), UL52 (primase) and UL8 (non-catalytic subunit). UL5 and UL52 subunits are functionally interdependent, and the UL8 component is required for the coordination of UL5 and UL52 activities proceeding in opposite directions with respect to the viral replication fork. Anti-viral compounds currently under development target the functions of UL5 and UL52. Here, we review the structural and functional properties of the UL5/UL8/UL52 complex and highlight the gaps in knowledge to be filled to facilitate molecular characterization of the structure and function of the helicase–primase complex for development of alternative anti-viral treatments.

Preservation of lagging strand integrity at sites of stalled replication by Pol α-primase and 9-1-1 complex

During genome duplication, the replication fork encounters a plethora of obstacles in the form of damaged bases, DNA–cross-linked proteins, and secondary structures. How cells protect DNA integrity at sites of stalled replication is currently unknown. Here, by engineering “primase deserts” into the Caenorhabditis elegans genome close to replication-impeding G-quadruplexes, we show that de novo DNA synthesis downstream of the blocked fork suppresses DNA loss. We next identify the pol α-primase complex to limit deletion mutagenesis, a conclusion substantiated by whole-genome analysis of animals carrying mutated POLA2/DIV-1. We subsequently identify a new role for the 9-1-1 checkpoint clamp in protecting Okazaki fragments from resection by EXO1. Together, our results provide a mechanistic model for controlling the fate of replication intermediates at sites of stalled replication.

Role of Amygdalin in Blocking DNA Replication in Breast Cancer In Vitro

Background: Amygdalin has anticancer benefits because of its active component, hydrocyanic acid. However, the underlying molecular mechanism is unclear. Objective: This study aimed to investigate the molecular mechanism by which amygdalin exerts antiproliferative effects in the human Michigan Cancer Foundation-7 (MCF-7) breast cancer cell line. Methods: MCF-7 cells were exposed to amygdalin at a particular IC50 value for 24 and 48 hours and compared to nontreated cells. An Affymetrix whole-transcript expression array was used to analyze the expression of 32 genes related to DNA replication. Results: Among the 32 genes, amygdalin downregulated the expression of 16 genes and 19 genes by >1.5-fold at 24 and 48 hours, respectively. At 24 hours, the downregulated genes from the DNA polymerase α-primase complex were POLA1, POLA2, PRIM1, and PRIM2; DNA polymerase δ complex: POLD3; DNA polymerase complex: POLE4, minichromosome maintenance protein (MCM) complex (helicase): MCM2, MCM3, MCM4, MCM6, and MCM7; clamp and clamp loader: PCNA; nuclease: FEN1; and DNA ligase: LIG1. At 48 hours, the downregulated genes from the DNA polymerase α-primase complex were POLA1, POLA2, and PRIM1; DNA polymerase δ complex: POLD3; DNA polymerase complex: POLE and POLE2; MCM complex (helicase): MCM2, MCM3, MCM4, MCM5, MCM6, and MCM7; clamp and clamp loader: PCNA, RFC2, and RFC3; RNase H: RNASEH2A; nucleases: DNA2 and FEN1; and DNA ligase: LIG1. Conclusion: Amygdalin treatment caused downregulation of several genes that play critical roles in DNA replication in the MCF-7 cell line. Thus, it might be useful as an anticancer agent.

ATR repression at telomeres by POT1a and POT1b: RPA exclusion and interference by CST

ABSTRACTTelomeres carry a constitutive 3’ overhang that can bind RPA and activate ATR signaling. POT1a, a single-stranded (ss) DNA binding protein in mouse shelterin, has been proposed to repress ATR signaling by preventing RPA binding. Repression of ATR at telomeres requires the TPP1/TIN2 mediated tethering of POT1a to the the rest of the shelterin complex situated on the ds telomeric DNA. The simplest version of the tethered exclusion model for ATR repression suggests that the only critical features of POT1a are its connection to shelterin and its binding to ss telomeric DNA binding. In agreement with the model, we show that a shelterin-tethered RPA70 mutant, lacking the ATR recruitment domain, is effective in repressing ATR signaling at telomeres. However, arguing against the simple tethered exclusion model, the nearly identical POT1b subunit of shelterin is much less proficient in ATR repression than POT1a. We now show that POT1b has the intrinsic ability to fully repress ATR but is prevented from doing so when bound to the CST/Polα/primase complex. The data establish that shelterin represses ATR with a tethered ssDNA-binding domain that excludes RPA from the 3’ overhang and suggest that ATR repression does not require the interaction of POT1 with the 3’ end or G4 DNA.